Microfluidic Isolation of Tumor Cells or Other Rare Cells from Whole Blood or Other Liquids

ABSTRACT

Microdevices are disclosed to efficiently, accurately, and rapidly isolate and enumerate rare cells, such as circulating tumor cells, from liquids such as whole blood, The system employs multiple parallel meandering channels having a width on the order of 1-2 cell diameters. The microdevices can be produced at low-cost, may readily be automated, and in many instances may be used without pre-processing of the sample. They may he used to isolate and enumerate rare cells, including for example the detection and diagnosis of cancers, cancer staging, or evaluating the effectiveness of a therapeutic intervention, or detecting pathogenic bacteria, The device may optionally he used to nondestructively capture and later to release target cells.

This application is a divisional of U.S. patent application No.12/992,225, filed Dec. 7, 2011, which is the National Stage ofInternational Application No. PCT/US2009/043697, filed May 13, 2009,which claims the benefit of and right of priority to U.S. ProvisionalPatent Application No. 61/053,727, filed May 16, 2008, the fulldisclosure of which is hereby incorporated by reference.

This invention was made with government support under grantIR33-CA099246-01 awarded by the National Cancer Institute of theNational Institutes of Health. The government has certain rights in theinvention. This invention was made with government support under grantEPS-0346411 awarded by the National Science Foundation. The governmenthas certain rights in the invention.

TECTINICA FIELD

This invention pertains to the isolation of tumor cells or other rarecells from whole blood or other liquids.

BACKGROUND ART

There is an unfilled need for improved techniques to isolate and detectcirculating tumor cells and other rare cells.

Most cancer-related mortalities result from metastasis. Cancer cells canbe transported from the primary tumor by the circulatory system or bonemarrow; some of these circulating cells may have metastatic potential.The ability to identify and count circulating tumor cells (CTCs) wouldbe enormously helpful in the diagnosis and prognosis of many types ofcancer. It has proven difficult, however, to reliably detect CTCs due totheir extremely low concentration among a high background of “spectator”cells in peripheral blood (e.g., red and white blood cells). It has beenreported that the concentration of CTCs in the blood correlates withmean survival time for breast cancer patients. It would be very usefulclinically, for example, to be able to accurately count 0-10 CTCs in 1mL of whole blood in a background of ˜10⁹ erythrocytes and ˜10⁶leukocytes.

In sampling rare events from a large population, three important metricsare: (1) throughput, the number of cells identified or the number ofsorting steps per unit time; (2) recovery, the fraction of the targetcells successfully retrieved from the input sample; and (3) purity, thedegree to which the recovered cells are free from “interfering” cells.In addition to these three metrics, the enriched cells must also becounted accurately.

Prior approaches to enriching CTCs in clinical samples have generallyproduced low recoveries with high purity, or low purity with highrecovery. In a handful of cases, both high purity and high recovery havebeen reported, but only with highly specialized sample processing andhandling equipment and techniques. For example, antibody-coated,micron-sized magnetic particles have been used to enrich CTCs with highpurity, but only modest recoveries (˜70%). Polycarbonate membranes withvarying pore sizes (8-14 μm) have been used to filter cells by size fromrelatively large volumes of blood (9.0-18 mL), with recovery of ˜85% ofthe CTCs, but at low purity due to the retention of large numbers ofleukocytes as well.

Another approach has been to use quantitative PCR; or to usereverse-transcription PCR to assay mRNA as a surrogate for CTCs. RT-PCRcan detect one CTC in an excess of 10⁶ mononucleated cells. However,RT-PCR assays are prone to high inter-laboratory variability, arenotoriously subject to false positives from environmental contamination,and require extensive sample handling and manipulation. Also, PCRtechniques will generally destroy the cells being sampled.

Among the difficulties encountered by the existing methods for isolatingand counting CTCs are one or more of the following: the need to selectrare CTC cells from the mononucleated fraction of whole blood, whichtypically involves the use of density gradient centrifugation to removethe far more numerous RBC's; the need for flow cytometry apparatus; orthe need for fluorescence microscopy. In addition to their cost andcomplexity, these procedures entail sample handling and transfer stepsthat can result in cell loss or contamination, which can dramaticallyaffect results, particularly when one is dealing with a very low numberof target cells to begin with.

Microfluidic systems can be used to process samples, including clinicalsamples, so as to minimize sample contamination and loss. However,microfluidic systems have not previously been widely used to processrelatively large sample volumes (e.g., 1 mL) due to the small dimensionsof the devices. For example, to fully process a 1.0 mL sample volumeusing a 30 μm×30 μm microchannel at a linear velocity of 1.0 mm s⁻¹would require ˜309 h (˜13 days). One approach has been to prepare ahigh-surface area immunological capture bed filled with microposts(e.g., ˜100 μm diameter×˜100 μm tall).

Patent application publication no. US2007/0026413A1 discloses a devicewith an array of obstacles (e.g., microposts) that relies on thehydrodynamics of flow through gaps between the obstacles, as well asspatial offsets between adjacent rows of the obstacles, and antibodieson the micropost surfaces to preferentially sort cell types by size,shape, chemical composition, or deformability. See also S. Nagrath etal., “Isolation of rare circulating tumour cells in cancer patients bymicrochip technology,” Nature, vol. 450, pp. 1235-1239 (2007); andpatent application publication nos. US2007/0264675A1; US2007/0172903A1;US2008/0124721A1 and US2006/0134599A1.

Patent application publication no. US2008/0318324A1 disclosesmicro-fabricated or nano-fabricated devices for separating,concentrating, and isolating circulating tumor cells or other particles.Fluidic channels having particular cross-sectional shapes or otherfeatures could be used for dispersion, distribution, or partition of thefluidic flow, in order to reduce the direct impact of cells againstexclusion features. The disclosure describes the use of so-called“effusive filtration”—redirecting, partitioning, dampening, ordispersing fluid flow to reduce physical impact on cells, while stillallowing filtration through apertures. In some cases curvature might beincluded as a feature of the filtration perimeter. The channel surfacescould be treated with anticoagulant compounds, compounds thatpreferentially bind to circulating tumor cells, or compounds thatprevent the sticking of cells.

P. Sethu et at., “Continuous flow microfluidic device for rapiderythrocyte lysis,”Anal. Chem., vol. 76, pp. 6247-6253 (2004) disclosesa microfluidic device for erythrocyte removal from a blood sample byselectively lysing the erythrocytes. Whole blood was mixed with a lysisbuffer, and the mixture was transported through a rectangular-wavemicrofluidic reaction channel.

M. Toner et al., “Blood-on-a-Chip,” Annu. Rev. Biomed. Eng., vol. 7, pp.77-103 (2005) provides a review of research concerning the use ofmicrodevices for manipulating blood and blood cells at the micro-scale.

Antibodies are not the only molecular recognition elements with highspecificity for selected target molecules. Aptamers are another exampleof recognition elements that can have high specificity. Aptamers aresingle-stranded nucleic acid oligomers with specific affinity for amolecular target, generally via interactions other than classicalWatson-Crick base pairing. In comparison to antibodies, aptamers(generally) have lower molecular weight and higher stability duringlong-term storage. Automated techniques are known in the art forselecting and synthesizing aptamers having specificity against a desiredtarget, e.g., a membrane protein. Aptamers may readily he immobilized onsolid support surfaces such as glass, polymers, or gold.

S. Lupold et al., Cancer Res., vol. 62, pp. 4029-4033 (2002) disclosedRNA aptamers directed against PSMA, and the use of those aptamers totarget lymph node metastasis prostate cancer (LNCaP) cells.

J. Phillips et al., Anal. Chem., vol. 81, pp. 1033-1039 (2009) disclosedthe use of aptamers on PDMS microchannel walls for the selection ofleukemia cells seeded (˜1×10⁶ cells/mL) in an aqueous buffer that wasalso loaded with non-cancerous cells.

A. Adams et cit., “Low abundant biomarker screening inpoly(methylmethacrylate) high aspect ratio microstructures usingimmunoaffinity -based molecular recognition,” Special Publication: RoyalSociety of Chemistry—Miniaturized Total Analysis Systems, vol. 1, pp.132-134 (2004) disclosed a PMMA high aspect ratio, antibody-decorated,microfluidic device to pre-concentrate low abundant cancer cells fromsuspensions of simulated blood. An optimum flow rate for this device wasfound to be 2 mm/s. Antibodies were attached to a carboxylated polymersurface, generated by exposure to UV radiation. The device had 17straight channels “with extreme rectangular character i.e. narrow (30-50um) and tall (250 μm)” to maximize collisions between cells and channelwalls. A capture efficiency of 1% was reported for a 50 μm channel, and100% for a 20 μm channel.

DISCLOSURETHE INVENTION

We have discovered a system to efficiently, accurately, and rapidlyisolate and enumerate rare cells, such as circulating tumor cells, fromliquids such as whole blood. It is now possible to exhaustively andrapidly interrogate large volumes (e.g., 1.0 mL or more) of unprocessedwhole blood for rare cells, such as CTCs. Rare cells have successfullybeen isolated and detected in prototype experiments when the backgroundcells outnumbered target cells by eight orders of magnitude; and evenhigher levels of sensitivity should be possible.

In a prototype embodiment of the novel high-throughput microsamplingunit (HTMSU), we have designed and successfully tested exceedinglyefficient, high-aspect ratio capture beds decorated with monoclonalantibodies (mABs) or aptamers specific for CTC membrane proteins. TheHTMSU has parallel, meandering (preferably sinusoidal orquasi-sinusoidal) fluid channels with a width on the order of 1-2 celldiameters. A preferred embodiment employs a label-free, highly specificconductivity sensor for the non-destructive detection of single cells.Tumor cells often have higher electrical conductivities than normalcells, for example, due to over-expression of charged membrane proteinsor glycoproteins, such as those with sialic acid molecules. Thisdifference lends itself to detection by conductivity measurements. Apreferred method for attaching capture elements is the photoresist-freemicropatteming technique disclosed in United States patent applicationpublication no. US2007/0191703A1. Using this technique the captureelements can be selectively attached to just the channel walls, withoutalso being attached to other portions of the device. Selectivelyattaching the capture elements to only the channel walls is preferred,and helps promote high recovery of the target cells. By contrast,attaching capture elements outside the channels could cause cells toanchor to spots in “unswept void volumes” that might not later bereleased; while not preferred in general, in sonic specific applicationsit could be useful also to attach capture elements to other surfaceswithin the device, surfaces outside the channels themselves. Thehydrodynamics are generally better-tuned within the capture channelsthan in the other areas of the device, and it is therefore preferred tocapture the cells only within the channels. We note that the side wallsof the channels can be exposed with a conventional mask and UVirradiation, because the light is typically not fully collinear. Thereare divergent light rays that hit the side walls. Also, there will bescattering that causes the side walls to be exposed to the UV radiation.

Devices in accordance with the present invention can be produced atlow-cost using micro-replication and fabrication technologies that areotherwise known in the art. The devices may readily be automated, and inmany instances may be used without requiring any pre-processing of thesample. The novel system may be used in many situations where it isdesirable to isolate and enumerate rare cells, including for example thedetection and diagnosis of cancers, cancer staging, evaluating theeffectiveness of a therapeutic intervention, or detecting pathogenicbacteria (e.g., in food or in environmental samples).

The HTMSU system is flexible, and may accommodate a wide variety ofmolecular recognition elements (antibodies, aptamers, etc.) to targetparticular types of rare cells. As one example, the channel walls couldbe decorated with monoclonal antibodies directed against E. coil O157:H7to detect that pathogenic bacterial strain at extremely lowconcentrations.

Preferred embodiments of the novel system employ one or more of thefollowing features or characteristics: “walled-in” channels rather thanposts are used for fluid flow and cell capture; posts, although notpreferred, may optionally also be present, but not to the exclusion of“walled-in” channels; multiple parallel channels are preferred toenhance throughput; sinusoidal, quasi-sinusoidal, or other meanderingchannel shape is used to enhance contact between cells in the fluid andthe channel walls; a very high capture efficiency is obtained; thechannels have a high aspect ratio (3:1 or more); the channel widthshould be on the order of 1-2 cell diameters; there is a uniform ornear-uniform pressure drop across the multiple channels; the devicematerial chosen to inhibit non-specific binding, PMMA is often usefulfor this purpose; highly-specific capture elements are used, e.g.,monoclonal antibodies or aptamers; the device is readily scalable bychanging the channel depth, the number of channels, or both; and thedevice may be used to nondestructively capture and later to releasetarget cells.

Recovery efficiencies of rare cells can be very high: 80%+, 85%+, 95%+,97%+. These rates are superior to any that have previously been reportedwith other devices or systems. Additionally, the rate of false positivescan be very low, less than 1 per 10 mL of sample. A low rate of falsepositives can be achieved by incorporating several independent modes ofspecificity into the device and its operation; preferred embodimentsemploy molecular recognition (e.g., monoclonal antibody), shear force toremove non-target cells that may be non-selectively adhering to thesurface, and the use of a detector that is selective for cells havingthe same size as the target cells. The invention may be used to recoverrare cells from a variety of liquids, including whole blood, water,urine, saliva, CSF, and others.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts schematically a prototype HTMSU in accordance with thepresent invention.

FIGS. 2A-D are histograms depicting the observed radial position of CTCsin different shaped microchannels at different velocities.

FIGS. 3A-C depict the results of various conductance measurements withthe novel system, using MCF-7 breast cancer cells and whole blood.

FIGS. 4A & B depict the results of various conductance measurements withthe novel system, using LNCaP prostate cancer cells and whole blood.

FIG. 5 depicts schematically the immobilization of cells on the surfaceof a channel in accordance with one embodiment of the invention.

FIGS. 6a and 6b depicts a cross-sectional view of two differentexemplary channels in accordance with embodiments of the invention,wherein the height of the channel 11 is at least about three times thewidth of the channel 12.

FIG. 7 depicts data showing the capture efficiency of CTCs in spikedwhole blood samples as a function of the cells' translational velocityusing 35 (down triangles, sinusoid; circles, straight), and 50 μm (uptriangles) wide microchannels. The microtluidic device consisted of asingle channel with the appropriate width and a depth of 150 μm.

MODES FOR CARRYING OUT THE INVENTION EXAMPLES 1-21 Fabrication ofPrototype HTMSU, and Isolation and Detection of Low Abundance MCF-7Breast Cancer Cells in Whole Blood Materials and Methods

EXAMPLE 1

HTMSU fabrication. FIG. 1 depicts schematically a prototype HTMSU inaccordance with the present invention. The prototype HTMSU has beensuccessfully fabricated and tested. The prototype device contained 51high-aspect-ratio, sinusoidal, parallel channels that shared a commoninput port and a common output port. Devices were replicated from amaster mold using hot embossing techniques that are otherwise known inthe art. See A. Adams et at., “Highly efficient circulating tumor cellisolation from whole blood and label-free enumeration usingpolymer-based microfluidics with an integrated conductivity sensor,” J.Am. Chem. Soc., vol. 130, pp. 8633-8641 (2008), and the supportingmaterial that is available at pubs.acs.org for additional detailsconcerning the fabrication of the master mold and the micro-replicationprocedures, the complete disclosures of which are incorporated byreference. The substrate used in the prototype HTMSU was poly(methylmethacrylate) (PMMA), which was chosen for its high fidelity inreplicating high-aspect ratio microstructures, its minimal non-specificadsorption of whole blood components, and the ability to functionalizethe surface of PMMA with different moieties through UV irradiation.

EXAMPLE 2

Antibody immobilization. Antibodies were immobilized in a two-stepprocess. First, the UV-surface-modified HTMSU device was loaded with asolution containing 4.0 mg/mL of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), 6.0 mg/mL of N-hydroxysuccinimide(NHS) in 150 mM 2-(4-morpholino)-ethane sulfonic acid at pH=6 (MES,Fisher Biotech, Fair Lawn, N.J.); and buffered saline (Sigma-Aldrich,St. Louis, Mo.) for 1.0 hr to form a succinimnidyl ester intermediate.The EDC/NHS solution within the device was then hydrodynamicallyreplaced with a 1.0 mg/mL solution of monoclonal anti-EpCAM antibody(R&D Systems Inc., Minneapolis, Minn.) in 150 mM PBS at pH=7.4(Sigma-Aldrich, St Louis, Mo.), which was allowed to react for 4 hours.The device was then rinsed with a solution of PBS (pH=7.4) to remove anyunbound anti-EpCAM antibodies. For additional details concerning theseprocedures, see the Adams el al. Supporting Information (2008), herebyincorporated by reference.

EXAMPLE 3

Apparatus. A PHD2000 syringe pump (Harvard Apparatus, Holliston, MA) wasused to hydrodynamically process samples in the prototype HTMSU. A luerlock syringe (Hamilton, Reno, Nev.) was fitted with a luer-to-capillaryadapter (Innova uartz, Phoenix, Ariz.) to connect the HTMSU and thepump. The flow rate of the syringe pump was programmable. The linearvelocities were calculated, based on the ratio of the cross-sectionalarea of the HTMSU capture channels to the programmed volumetric flowrate. The flow rates were confirmed by tracking suspended cells over afixed 80 μm region via optical microscopy.

The prototype HTMSU was fixed to a programmable, motorized stage of anAxiovert 200M (Carl Zeiss, Thomwood, N.Y.) microscope, which couldmonitor cells in the HTMSU by fluorescence or bright field imaging.Videos of cell transport were captured at 30 frames per second using amonochrome CCD (JAI CV252, San Jose. Calif.). For fluorescenceobservation of MCF-7 cells, dyes were excited with a Xe arc lamp anddye-specific filter sets (Carl Zeiss, Thornwood, N.Y.). Each filter cubecontained a dichroic mirror, emission filter, and excitation filter.

EXAMPLE 4

Integrated conductivity sensor. The conductivity electrodes were Ptwires (˜75 μm) in guide channels embossed into the fluidic network andpositioned orthogonal to the fluid output channel. The Pt wire wasinserted into the guide channels prior to thermal bonding of the coverplate to the substrate. Once the wire was positioned, the substrate/wireassembly was placed between glass plates and clamped together and heatedto slightly above the glass transition temperature of PMMA, to embed thewire into the guide channels. The wire spanned the entire depth of theoutput channel. The wire was then cut to form an electrode pair, using ahigh precision micromilling machine (KERN MMP 2522, KERN Micro-undFeinwerktechnik GmbH & Co. Eschenlohe Germany) with a 50 μm bit.Following machining of the Pt wire and UV activation of the channelpolymer surfaces, the cover plate was aligned with the embossedsubstrate via alignment marks, and the assembly was clamped between twoglass plates and heated to bond the components to one another,

For further details concerning the preferred conductivity sensor, see D.Patterson, “Conductivity Counter,” U.S. patent application Ser. No.12/388,904, filed Feb. 19, 2009; and the Adams et al. supportingmaterial (2008); the complete disclosures of both of which areincorporated by reference.

Conductivity was measured in TRIS-glycine buffer containing 0.25% (w/w)trypsin, 0.18 mM TRIS, 47 mM glycine, and 0.05% (v/v) Tween-20,sometimes referred to as the “CTC-releasing buffer.” The CTC-releasingbuffer had a relatively low conductivity (˜50 μS/cm, pH 7.2). Thetrypsin in the CTC-releasing buffer acts to remove bound cells (CTCs)from the capture channel surface.

EXAMPLE 5

Cell suspensions. Citrated whole rabbit blood was purchased fromColorado Serum Company (Denver, Colo.). (The blood contained 10% (w/w)sodium citrate to inhibit coagulation.) The MCF-7 cells (a breast cancercell line), growth medium, phosphate buffered saline, trypsin, and fetalbovine serum were purchased from the American Type Culture Collection(Manassas, Va.). Adherent MCF-7 cells were cultured to 80% confluence inDulbecco's modified Eagle's Medium supplemented with high glucose, andcontaining 1.5 g L⁻¹ sodium bicarbonate (NaHCO₃), 15 mM HEPES buffer,and 10% fetal bovine serum (FBS).

Some of the MCF-7 cells were stained for fluorescence visualizationexperiments with PKH67, a fluorescein derivative that contains alipophilic membrane linker (Sigma-Aldrich, St. Louis, Mo.). Themanufacturer's suggested protocol for cell staining was modified bydoubling the dye concentration, so that the fluorescent labels were moreevenly distributed over the cell membranes. The cell counts in wholeblood seeding experiments were determined by counting three aliquots ofcells with a hemocytometer. The cell count accuracy was ±10%.

Results and Discaussion (N CF-7 Cell Experiments)

Model CTC system. We used the MCF-7 cell line as a model for CTCselection and enumeration with the prototype HTMSU. MCF-7 is a breastcancer cell line that over-expresses the membrane-bound moleculeepithelial cell adhesion molecule (EpCAM). MCF-7 cells are ˜15-30 μm indiameter (mean=24 μm). They have been closely associated withmicro-metastatic breast cancer. MCF-7 membranes have an average of˜5.1×10⁵ EpCAM molecules per cell. Monoclonal antibodies for EpCAM arecommercially available.

We found several experimental and device parameters that affected theperformance of the HTMSU We have taken preliminary steps towardsoptimizing these parameters to enhance performance of the HMISU. Amongthese parameters are the following: (1) Throughput—linear flow velocity,pressure drop, processing time; (2) recovery—capture channel geometry(shape and width), cell flow dynamics; (3) purity—surface design tominimize non-specific adsorption and to provide high selectivity fortarget cells.

EXAMPLE 6

Pressure drop. A primary goal for the prototype HTMSU was to processwhole blood directly in a reasonable time. We designed high-aspect ratiocapture channels to enhance throughput. (Aspect ratio=channelheight/channel width.) If the channel width and height were both on theorder of the cell dimensions (aspect ratio≈1), then the capture of evena single cell within a channel would cause a large pressure drop, andcould possibly damage the captured cell. Other potential sources ofobstruction also become more likely when both the width and the heightare small. For the prototype device we chose a capture channel with anaspect ratio of 43:35 μm wide by 150 μm deep. These dimensions mayreadily be reproduced by hot embossing techniques otherwise known in theart. Assuming a blood viscosity of 4.8 cP (hematocrit=0.4), wecalculated that the pressure drop for a channel 35 μm×35 μm (L=3.5 cm)would be ˜7.4×10³ Pa. while the pressure drop for a 35 μm×150 μm channelwould be ˜2.9×10³ Pa, a reduction of more than 60%.

EXAMPLES 7-10

Flow dynamics. When a capillary has dimensions more than ˜15% largerthan the dimensions of the cells being transported, the cells tend tomigrate toward the central axis of the tube, and to leave a cell-freelayer adjacent to the capillary wall, typically ˜4 μm thick. Because thecapture elements (e.g., antibodies) are tethered to the channel wall,this “focusing” away from the wall tends to reduce the number ofencounters between target cells (e.g., CTCs) and the recognitionelements. To investigate this phenomenon quantitatively, we stained CTCswith a membrane-specific fluorescein derivative, and imaged them as theywere transported through unmodified (no mAB), 35 μm-wide microchannelswith either a straight or a sinusoidal configuration. The results ofthese experiments are shown in FIGS. 2A-D

FIGS. 2A-D are histograms depicting the observed radial position of CTCsin microchannels at linear velocities (U) of 1.0 mm/s (FIGS. 24 and C)and 10 mm/s (FIGS. 2B and D); in straight (FIGS. 24 and B) andsinusoidal (FIGS. 2C and D) channels. The central lines represent themicrochannel's central axis. The cells were stained with a fluoresceinlipophilic membrane dye, PKH67, and were imaged by fluorescencemicroscopy.

The straight channels (FIGS. 2A and B) displayed a cell-free zone,similar to what has been observed in straight capillaries. The thicknessof this cell-free zone increased with increased velocity U. The observedflow dynamics were quite different in the sinusoidally-shaped channels(FIGS. 2C and D). First, no marginal zone appeared along the edge of themicrochannel wall, and second, the radial distribution of cells seemedto be largely independent of changes in U. Thus more efficient captureof cells results when sinusoidal (or quasi-sinusoidal, or othermeandering) channels are used even when relatively high linearvelocities are used to process large input volumes over relatively shorttimes. Without wishing to be bound by this hypothesis, we believe thatthe cells migrate to the outside of the curved channel primarily due totwo factors: (1) a cross-stream velocity component due to the reversalof the direction of curvature, which promotes re-circulation and mixing;and (2) centrifugal force acting on the cells, which tends to “push”cells toward the outer walls.

We also calculated the Reynolds number and the Dean number for the 35μm, curved channels. The Dean number is a dimensionless quantity thataccounts for both the radius of curvature and the hydraulic diameter ofa curved channel. For the sinusoidal channels used in our prototypedevices, the Dean number was calculated to be ˜1.1 at a translationalvelocity of 10 mm s⁻¹, well below the threshold value of ˜36 at whichthe flow becomes unstable.

EXAMPLES 11-14

Effects of channel width and linear velocity on capture efficiency. Weexamined the effects of varying the dimensions and shape of the capturechannels, and the linear flow velocity. We measured CTC captureefficiency in straight channels 20, 35, and 50 μm wide, and in a 35μm-wide sinusoidal channel, at various flow rates. The results of thesestudies are depicted in FIG. 7 as plots of CTC capture efficiency (5)versus linear velocity, U (mm s⁻¹).

The 20 μm-wide, straight channel trapped essentially 100% of the targetcells before entering the channel, at all flow rates investigated.Observation by fluorescence microscopy revealed that most cells did noteven enter the capture channel, because the CTCs were typically widerthan the 20 μm channel. For those cells that did enter the channels, thecell membranes were in constant proximity to the antibody-coated channelwalls, and were typically captured within the first 1-2 mm of the 3.5cm-long channel. The 20 μm device consistently failed from microchannelblockage in these tests, and we consider it to be too narrow to be usedfor many cell types (although such a width or even narrower might bepractical for capturing smaller cells, such as bacteria), Furthermore,at higher linear flow velocities blockages can lead to unacceptable headpressures, For these reasons it is preferred to use a channel width thatis at least as large as the diameter of the average target cell.

We also observed that for all channel widths above 20 μm, in both linearand sinusoidal channels, the cell capture efficiency reached a maximumat a linear flow velocity ˜2 mm s⁻¹. Capture efficiency declined at bothfaster and slower flow rates. Capture efficiency was higher for narrowerchannel widths, provided that the channel was not so narrow that it wasreadily susceptible to blockage. The highest capture efficiency wasobtained with the sinusoidal-shaped, 35 μm-wide channel (˜97%). Thisoptimal flow velocity will vary, depending on the particular type ofcell targeted, and the particular capture element used.

A device with a single 35×150 μm channel, operated at a linear flow rateof ˜2 mm s⁻¹, would process 1 mL of fluid in ˜9.5×10⁵ s (˜26 h).Increasing the linear flow rate would decrease capture efficiency, so weinstead designed a prototype device with multiple capture channels (allof similar dimensions), having a single, common input and a single,common output. The prototype device had 51 capture channels, reducingthe processing time for 1 mL of fluid to ˜1900 s (˜31 min). Using acommon output for all the capture channels allowed for simple collectionand pooling of the selected cells. The 51-channel prototype device isdepicted in FIG. 1. If the channel depth were increased to 250 μm (anaspect ratio of 7.14 for a 35 μm-wide channel), and if the number ofparallel channels were doubled, then the sampling time for this samevolume input could be reduced to ˜2.7 min. Also, larger sample volumes(e.g., 10 mL or more) could be processed in a reasonable time.

EXAMPLES 15-16

Shear effects on captured cells. We also evaluated shear forces oncaptured cells, to determine whether flow-induced shear could eitherdetach the cells from the walls or damage the cells. Using a simplemodel of the forces involved, we calculated that the linear flowvelocity required to detach the EpCAM-expressing cells from a PMMA walldecorated with anti-EpCAM antibodies would be on the order of 10² to 10⁴cm s⁻¹, depending on the degree to which the captured cells wereflattened and elongated on the antibody-decorated surface. This range ofvelocities is substantially greater than the flow rates used in ourexperiments, implying that shear forces should not be expected topresent difficulties. We observed several captured cells continuouslyduring tests employing linear velocities up to 10.0 cm s⁻¹, neither celldamage nor disruption of cell-wall adhesion was seen.

EXAMPLE 17

Detaching and counting intact cells. After cells have been captured, itwill usually be desirable to selectively release the captured cells at alater time without damaging them, for example to count or otherwisecharacterize them. There are strong adhesion forces in a typicalantigen-antibody system, so an enzymatic or other selective releasingmechanism should be used. For example, we have successfully used theproteolytic enzyme trypsin to release captured cells undamaged.Proteolytic digestion and release of the captured cells typicallyrequired less than 10 minutes.

EXAMPLES 18-20

Conductivity sensor for cell enumeration. Two integrated Pt electrodes,50 μm apart, were used in a conductivity sensor to count cells. Thecharacteristic parameter K (the ratio of the electrode gap to theelectrode area) was chosen as ˜0.01 μm⁻¹ to detect larger, target CTCspreferentially over smaller leukocytes or erythrocytes that might stillbe present in small numbers as the result of non-specific adsorption.

The capture channels of the prototype HTMSU were tapered from 150 μm toa depth of ˜80 μm as they approached the conductivity sensor, in orderto match the Pt electrode diameter and to have a sampling efficiencynear 100%. FIGS. 3A-C depict the results of various conductancemeasurements. FIGS. 3A and B depict conductance responses, in arbitraryunits (AU). FIG. 3A depicts the response of the electrodes to testsamples spiked with leukocytes (lower curve) or erythrocytes (uppercurve). In both cases the cell density was 150 cells/μL, in TRIS-Glycinebuffer, transported through the integrated conductivity sensor at a flowrate of 0.05 μL/min. We observed that the electrodes were essentiallyinsensitive to both leukocytes and erythrocytes. Briefly: theconductivity sensor is sensitive to changes in the local bulk solutionconductance. When a single cell traverses the electrode pair, a changein the measured local bulk conductivity can result. RBCs produced noappreciable signal, simply as a result of their small size. Theconductance of WBCs is similar to that of the carrier buffer, resultingin no apparent signal. However, the conductance of CTCs will generallydiffer from that of the buffer, allowing them to be selectivelydetected.

The chemical composition of CTCs makes their electrical propertiesdistinct from those of erythrocytes or leukocytes. For example, CTCsgenerally have a low membrane potential and a low impedance. Also,cancer cell membranes generally have higher numbers ofnegatively-charged sialic acid molecules. Due to the differentialbetween the conductance of CTCs and that of the carrier buffer, and dueto the cells' relatively large size, distinct signals were recorded bythe conductivity sensor, corresponding to single CTCs.

We then seeded 1 mL of whole blood with 10±1 CTCs, pumped the samplethrough the prototype HTMSU at 2.0 mm/s to concentrate the CTCs into avolume ˜190 nL, released the captured cells with trypsin, and countedthem with the conductivity sensor at a volumetric flow rate of 0.05μL/min. See FIG. 3B, showing the measured conductivity data using a3-point Savitsky-Golay numerical smoothing filter. At a. signal-to-noisethreshold of 3:1 (=99.7% confidence level), there were 10 peaks in theconductance trace that we assigned to CTCs (indicated with asterisks).Only positive conductance spikes (relative to background) were scored asCTCs, not negative spikes. The “middle” or “baseline” curve also shownin FIG. 3B corresponds to a control sample of whole blood that lackedMCF-7 cells, but that had also been processed through the HTMSU. The CTCscoring system was verified through bright field microscopy, whichconfirmed that individual CTCs were correlated with positive signalsrelative to the background conductance, and that the negative signalsappeared to be correlated with non-cellular particulates. The disparityin the magnitudes of the CTC peaks evidently arose from differences incell morphology and composition, perhaps due at least in part tovariations in mitotic phase. With the control blood sample (not spikedwith CTCs) no conductance signals exceeded the 3σ criteria, i.e.. weachieved a false positive rate of 0 in this particular test.

EXAMPLE 21

Further testing of the conductivity sensor for cell enumeration. Tofurther verify the recovery and detection efficiency of the system, weconducted further tests in which the number of seeded CTCs varied over abroad, physiologically-relevant range (10-250 CTCs per mL of wholeblood). See FIG. 3C. The best-fit line for the measured data had a slopeof 0.945 with an intercept near 0 (r²=0.9988), indicating an overallrecovery and detection rate of ˜95% for the CTCs, with a low falsenegative rate, and a very low false positive rate.

EXAMPLES 22-31 Using the Prototype HTMSU to Isolate and Detect LowAbundance LNCaP Prostate Cancer Cells in Whole Blood

Prostate tumor cells over-express prostate specific membrane antigen(PSMA). PSMA can be used as a marker to select low-abundance prostatetumor cells from highly heterogeneous clinical samples, including wholeblood. We have developed an HTMSU system employing aptamers tospecifically bind PSMA. The surface density of the PSMA-specificaptamers on the PMMA surface was ˜8.4×10¹² molecules/cm². At a linearvelocity of 2.5 mm/s, we recovered ˜90% of prostate cancer cells from awhole blood sample. Captured cells were subsequently released intactfrom the surface using 0.25% (w/v) trypsin. The HTMSU device used inthese experiments was generally similar to the prototype devicedescribed in Examples 1, 3, 4, & 6-14, and depicted in FIG. 1, except asotherwise stated. Neither pre-processing of the blood, nor staining ofthe cells was required. Nuclease-stabilized, in vitro-generated RNAaptamers were immobilized onto UV-modified sinusoidal capture channelsin the HMISU using carbodiimide coupling chemistry and a linker toenhance accessibility of the surface-bound aptamer.

EXAMPLE 22

Reagents. The following reagents were purchased from Sigma-Aldrich (St.Louis, Mo.): reagent grade isopropyl alcohol,1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC),N-hydroxysuccinimide (NHS), fetal bovine serum (FBS) and2-(4-morpholino)-ethane sulfonic acid (MES). The nuclease-resistant RNAaptamer, (NH₂—(CH₂)₆—(OCH₂CH₂)₆- (ACCAAGACCUGACUUCUAACUAAGUCUACGUUCC)(SEQ ID NO. 1), was obtained from Eurogentec (San Diego, Calif.). Randomsequence oligonucleotides were obtained from Integrated DNA Technologies(Coralville, Iowa). Monoclonal anti-EpCAM antibody was obtained from R&DSystems Inc. (Minneapolis, Minn.). The LNCaP (prostate cancer cellline), MCF-7 (breast cancer cell line), growth media, HEPES buffer,phosphate buffered saline (PBS) and trypsin were all purchased fromAmerican Type Culture Collection (Manassas, Va.). Citrated rabbit bloodwas purchased from Colorado Serum Company (Denver, Colo.) Tris-Glycinebuffer was obtained from Bio Rad Laboratories (Hercules, Calif.). Allsolutions were prepared in nuclease-free water, purchased fromInvitrogen Corporation (Carlsbad, Calif.). Nuclease-free microfuge tubespurchased from Ambion Inc. (Foster City, Calif.) were used for thepreparation and storage of all samples and reagents. A fluoresceinderivative, PKH67, Which contains a lipophilic membrane linker for cellstaining, was purchased from Sigma-Aldrich (St. Louis, Mo.).

Example 23

Cell suspensions. LNCaP and MCF-7 cells were cultured to 80% confluencein Dulbecco's Modified Eagle's Medium, supplemented with high glucose,and containing 1.5 g/L sodium bicarbonate (NaHCO₃), 15 mM HEPES buffer,and 10% FBS. A 0.25% trypsin solution in 150 mM PBS was used to harvestthe LNCaP and MCF-7 cells from the culture plate. LNCaP and MCF-7 cellswere stained with PKH67 for fluorescence microscopy, at twice themanufacturer's recommended concentration of PKH67 to more evenlydistribute fluorescent labels over the cell surface. Cell counts forseeding experiments in whole blood were determined by counting threealiquots of cells using a hemocytometer. The cell count accuracy was±10%.

EXAMPLE 24

Irradiating channel surfaces and forming the HTMSU. The cleaned PMMAdevices and cover plates were exposed to ultraviolet (UV) radiationthrough a mask to form carboxylate moieties on the surface of thecapture bed region of the device (but not in other parts of the HTMSU).UV irradiation was transmitted through an aluminum mask for 10 min at 15mW cm⁻². The parts were then aligned and clamped together between twohorosilicate plates. The cover plate was thermally fused to thesubstrate in a convective oven. The temperature was increased from 50°C. to 101° C. at a rate of 20° C./train, and held for 15 min. at 101°C., a temperature that is slightly above the glass transitiontemperature of the UV-modified material. Polyimide-coated, fused silicacapillaries were then inserted into the inlet port of the HTMSU tointroduce samples into the device with a programmable syringe pump(Harvard, Holliston, Mass.).

EXAMPLE 25

Conductivity sensor. Pt electrodes (d=76 were placed in guide channelsorthogonal to the fluidic output channel. Insertion of the electrodeswas monitored with a microscope to carefully control the inter-electrodegap (50 μm). The cell constant of the conductivity sensor, K, was ˜0.01μm⁻¹, chosen to optimize the specific detection of UNCaP cells with anaverage diameter ˜25 μm with electrodes have a diameter ˜75 μm.

EXAMPLE 26

Antibody immobilization. Antibodies were immobilized in a two stepprocess. The UV-modified thermally assembled HTMSU device was loadedwith a solution containing 4 mg/mL EDC, 6 mg/mL NHS, and 150 mM MES(pH=6) for 1 h at room temperature to obtain a succinitnidyl esterintermediate. The EDC/NHS solution was then removed by flushingnuclease-free water through the device. Then an aliquot of 1.0 mg/mL ofmonoclonal anti-EpCAM antibody solution in 150 mM PBS (pH=7.4) wasintroduced into the HTMSU, and allowed to react for 4 h. The device wasthen rinsed with a solution of PBS (pH=7.4) to remove anynon-specifically bound antibody molecules.

EXAMPLE 27

Aptamer immobilization. In one embodiment we employed aptamers as thecapture element rather than antibodies. Among the advantages of aptamersare the ordered nature of their attachment to the solid surface (e.g.,via the 5′ end), rather than the more random locus of attachment withantibodies (e.g., via primary amine groups on the antibody); the abilityto carefully control the aptamer/surface distance to improveaccessibility; and the robust nature of the molecular recognitionelements. Compared to antibodies, aptamers are more easily stored forlonger times while maintaining activity. For example, unlike antibodiesaptamers may be stored at room temperature without substantial loss ofactivity.

FIG. 5 depicts this embodiment schematically. PMMA film 1, on theinterior of the channel, has UV-activated surface layer 2. Activatedsurface layer 2 is attached via covalent linkages 3 to aptamers 4. LNCaPcells 5 are pumped over the surface, where some of the cells 5 areimmobilized by binding to aptamers 4.

Aptamers were immobilized on PMMA surfaces in a single step. FollowingUV activation, the activated PMMA surfaces were incubated with asolution containing 10 μM either of the PSMA aptamer or of randomoligonucleotides, and allowed to incubate for 2-3 h at room temperature.Each oligonucleotide solution also contained 4 mg/mL EDC, and 6 mg/mLNHS in 150 mM MES (pH=6). For decorating planar PMMA films, the PMMAsurface was immersed in the reaction solution. Following reaction, thePMMA surface was rinsed with a solution of PBS (pH=7.4) to remove anynon-specifically bound constituents.

EXAMPLE 28

Determination of aptamer surface density. A clean surface plasmonresonance (SPR) gold sensor surface was coated with 300 _L of a PMMAsolution (1.0 mg of PMMA in 10 _L of CH₂Cl₂) in a custom built spincoater, and was spun at 1,500 rpm for 1 min. The PMMA film was thenUV-activated and aptamers were immobilized on the PMMA under conditionsotherwise identical to those described above. The SPR response wasmeasured after each treatment with a BIACORE X SPR instrument(Piscataway, N.J.) using DI water. The difference in SPR response beforeand after aptamer immobilization was used to estimate the number ofmolecules/cm' using the manufacturer's published conversion factors.

EXAMPLE 29

LNCaP cell capture. A luer lock syringe (Hamilton, Reno, Nev.) with aluer-to-capillary adapter (Inovaquartz, Phoenix, Ariz.) connected thepump to a capillary, which was in turn sealed to the input port of theHTMSU. A pre-capture rinse was conducted with 0.2 mL of 150 mM PBS at 50mm/s linear velocity to maintain isotonic conditions. Then the cellsuspension was pumped through the HTMSU at a selected velocity, followedby a post-capture rinse with 0.2 mL of 150 mM PBS at 50 mm/s to removeany non-specifically adsorbed cells. The test sample was 1.0 mL of wholeblood seeded with 20±1 LNCaP cells, and the control sample was anotherwise identical sample of whole blood without LNCaP cells. Theoptimal linear flow velocity of 2.5 mm/s was used.

In some instances the HTMSU was fixed onto a programmable motorizedstage of an Axiovert 200M microscope (Carl Zeiss, Thornwood, N.Y.),Video images were collected at 30 frames per second (fps) using amonochrome CCD (JAI CV252, San Jose, Calif.). A Xe are lamp was used toexcite the fluorescent dyes incorporated into the cell membranes.

EXAMPLE 30

Release of bound LNCaP cells from the HTMSU. Following thepost-cell-capture rinse with PBS, a 0.25% (w/v) trypsin solution inTris-Glycine buffer (pH=7.4) was pumped through the HTMSU. The capturedcells could be observed microscopically until they were released.

EXAMPLE 31

Counting the released cells by conductivity measurements. Released cellswere pumped at 0.05 μL/min through the Pt electrodes for counting.Tris-glycine buffer was selected as the major component in the releasebuffer due to its low conductance; i.e., it is preferred that the buffershould have a conductivity substantially different from that of thetarget cells, and in general that the buffer's conductivity should belower than that of the target cells. The conductance response depictedin FIG. 4A exhibited 18 peaks that we assigned to single LNCaP cells,based on a signal-to-noise threshold of 3 (99.7% confidence level). Theasterisks designate peaks that were identified as LNCaP cells. Thearrowheads represent non-LNCaP cell events. Again, only positive signalswere assigned to the target cells; negative spikes, with lowerconductance, are believed to have resulted from particulates. The 18peaks represented a recovery of ˜90% of the cells. The insets to FIG. 4Adepict magnified views to illustrate the 3:1 signal-to-noisediscrimination threshold. The data were smoothed by the Savitsky-Golaymethod (using a 25 point smoothing fwiction).

The lower curve in FIG. 4A depicts the conductivity measurement for thecontrol sample of whole blood without LNCaP cells. No single-cell spikeswere seen in the data trace, indicating that the spikes seen for theLNCR-seeded whole blood were indeed due to the tumor cells, and that thepurity of the LNCaP cell selection was close to 100% (i.e., very fewfalse positives).

FIG. 4B depicts a calibration plot of “conductivity enumeration” versusthe actual number of seeded LNCaP cells, over a range of 10-250 LNCaPcells per mL whole blood. The best-fit linear plot had a slope of 0.990,with an intercept near zero (r²=0.9997). Even at the lowest LNCaP cellload tested, the data still fit the linear function very well. Thus thenovel system is well-suited to detect circulating tumor cells in wholeblood, even at extremely low concentrations. In 1 mL of whole blood,there are typically about 2.5×10⁹ erythrocytes; thus, at the lowestLNCaP cell load investigated, the enrichment factor was approximately2.5×10⁸—a difference of eight orders of magnitude. At the optimized flowrate of 2.5 mm/s used in these experiments, the time required toexhaustively process 1 mL of blood was ˜29 min.

We observed negligible adhesion of LNCaP cells either to pristine PMMA,or to PMMA linked to random-sequence DNA oligonucleotides. The adhesionforces of the LNCaP cells to these surfaces were not strong enough towithstand the hydrodynamic shear from the laminar fluid flow. However,LNCaP cells were efficiently captured when the PSMA-specific aptamer wastethered to the channel walls.

When the flow velocity is too fast, the resulting decrease ininteraction time between cells and the capture elements reduces thenumber of potential binding events. When the flow is too slow, thereduced velocity leads to a decrease in the encounter rate between thecells and the immobilized recognition element. The optimal flow rate mayreadily be determined for a given combination of HTMSU configuration,capture element, and target cell type.

We observed maximum cell capture efficiency for LNCaP cells and aptamersat a translational velocity of ˜2.5 mm/s, under the conditions employedin this study. By contrast, we found the optimum linear translationalvelocity for the anti-EpCAM antibody system to be slightly lower, ˜2.0mm/s. It thus appeared that the reaction rate for the EpCAM-antibodyinteraction was slightly slower than the reaction rate for thePSMA-aptamer interaction.

We also examined the possibility of non-specific adsorption orrecognition of other CTC-types using the breast cancer cell line MCF-7as an example. No MCF-7 cells were seen when the PMMA capture beds weredecorated with the anti-PSMA aptamers, and an MCF-7-spiked sample ofwhole blood was processed as otherwise described above.

There are two principal biosynthetic forms of PSMA in the LNCaP cellmembrane: the mannose-rich PSMAM form, and the glycosylated PSMAc form.PSMAM is highly sensitive to trypsin, while PSMAc is trypsin-resistant.Because trypsin efficiently released the captured LNCaP cells, itappears that the PSMAM form predominated, or at least that itpredominated in attaching to the anti-PSMA aptamers.

Nearly 100% of cells had detached within 7 min after the trypsin hadbeen introduced. Microscopic bright field observation at the Ptelectrodes confirmed that the released cells appeared to be intact.

EXAMPLE 32 Using the Prototype IITMSU to Efficiently Remove Red BloodCells from Whole Blood EXAMPLE 32

In another embodiment of the invention, red blood cells (“RBCs” or“erythrocytes”) are efficiently removed from a 100 μL sample of wholeblood in less than 1 minute using our prototype HTMSU. Aptamers are usedto selectively bind the RBCs and clear them from the sample, leaving thewhite blood cells for further analysis or experimentation. Clearing theRBCs first may make it easier for the larger cells to migrate or diffuseto the channel walls and be captured. Advantages of using the presentinvention for clearing RBCs as compared to other methods such ascentrifugation (i.e., apheresis) is that the clearance can be completedin a very short time period, smaller volumes of blood can be processed,the separation produces purer fractions with a higher recovery ofnon-cleared cells, and the device can be directly integrated withmolecular analysis devices to genotype or otherwise characterize thenon-cleared platelet and leukocyte fractions.

In carrying out various molecular analyses and other tests on wholeblood, it is often necessary to deplete the sample of RBCs.Centrifugation has been the principal technique previously used toseparate fractions of blood, including RBCs, based on their densities.There have been relatively few prior reports of microfluidic techniquesfor depleting RBCs, and those reports have focused primarily on whiteblood cell isolation via morphological differences.

An HTMSU as otherwise described in the previous examples is used torapidly, efficiently, and selectively remove RBCs from samples of wholeblood. RBC-specific aptamers are covalently linked to the cell capturebed walls: In one embodiment the aptamer is5′-CGAATCGCATTGCCCAACGTTGCCCAAGATTCG-3′ (SEQ ID NO. 2), which has beenreported to bind specifically to an RBC membrane protein. See K. Morriset al., Proc. Natl Acad. Sci. USA, vol. 95, pp. 2902-2907 (1998). Anamino-terminal cross-linker attached to the aptamer (5′NH₂—(CH₂)₆—(CH₂CH₂O)₆-TTTTT- Aptamer) facilitates the formation of astable amide bond with UV-generated carboxyl groups on the PMMA surface.The K_(D) for the binding of this aptamer to the RBC membrane proteinthat it recognizes is ˜1.8 nM, and the number of binding sites on theouter membrane of one RBC is ˜6.5×10³/cell.

In preliminary tests the HTMSU decorated with RBC-specific aptamers hassuccessfully and efficiently removed essentially all RBCs from a 100 μLsample of whole blood within 1 minute (as determined on conventionalhemocytometry slides), while allowing recovery of essentially all WBCsand plasma. In a control HTMSU decorated with random DNA sequences,essentially no RBC clearance was observed.

Using other suitable aptamers antibodies), other components may beselectively enhanced or depleted from a sample, for example platelets,neutrophils, other white blood cells, and so forth.

Miscellaneous. EXAMPLE 33

Further uses of the invention. The invention has been described abovelargely with respect to embodiments for detecting and enumerating cancercells in blood. The invention is well-suited for other uses as well. Itmay be used for detecting and isolating other rare cells in blood orother liquids. For example, it may be used for detecting and isolatingfetal cells in maternal blood, or maternal cells in an infant or anadult, or pathogenic bacteria in a water supply, or for isolating stemcells, or other instances of rare cells. The invention may also be usedto clear target cells from a sample, e.g., to remove erythrocytes from atest sample, for example prior to screening mRNA from a blood sample; orto remove leukocytes from whole blood, platelets, or plasma prior totransfusion; or to remove tumor cells from bone marrow prior to a bonemarrow transplant.

Where applicable, non-specifically bound, potentially interfering cells,e.g., erythrocytes non-specifically adhering to a PMMA surface, canoften be dislodged by hydrodynamic shear, simply by increasing the fluidflow rate.

Definitions.

As used in the specification and claims, unless context clearlyindicates otherwise, the following terms should be understood to havethe following meanings:

Two or more channels are “parallel,” or fluid flow within two or morechannels is “parallel,” if the flow occurs (or can occur) in thechannels in unison. “Parallel” should be understood as contrasting with“serial” or in “series,” a meaning that is analogous to that used in theterminology of electrical circuits. “Parallel” channels may indeed be,but are not necessarily, parallel in the geometrical sense.

“Channels” are separated paths for fluid flow. Multiple channels, evenadjacent channels, are physically separated from one another, i.e., theyare not in fluidic communication with one another, along most or all oftheir length. For example, fluid pathways that are separated from oneanother by walls, such as depicted in FIG. 1, are considered to be“channels” within this definition; even if they may share a common inputor a common output, they are physically separated (not in fluidiccommunication) along most of their length. By contrast, a system inwhich fluid flow is defined by obstacles such as posts, in which fluidflow along different paths is interleaved and in which fluid paths arein communication with one another, is not considered to constitute“channels” within the scope of this definition, even if there might becertain preferred paths for fluid flow.

The “length” of a channel is the distance that fluid traverses whengoing through the channel, along the most direct route from one end tothe other. FIGS. 6a and 6b depicts a cross-sectional view of a channel.As shown in FIGS. 6a and 6 b, the “width” 12 and “height” 11 of achannel are characteristic dimensions of a cross-section of the channel,both taken in a direction perpendicular to the length of the channel,and also perpendicular to one another. The “width” is generally thesmaller of the two characteristic dimensions, and the “height” thelarger of the two. The use of the terms “length,” “width,” and “height”should not be construed to imply that the channel necessarily has anyparticular shape. The length of a channel will usually be substantiallylarger than either its width or its height. The “aspect ratio” of achannel is the ratio of its height to its width.

“Sinusoidal” refers to a standard mathematical sinusoid, e.g., one thatmight he written in the form

y(x)=A·sin(kx+θ)+D

“Quasi-sinusoidal” refers to a periodically oscillating channel that mayor may not have the shape of a standard sinusoid. As just one possibleexample, a series of consecutive semicircles, facing in alternatingdirections and joined to one another end-to-end, would be considered“quasi-sinusoidal.” “Sinusoidal” forms are thus a subset of“quasi-sinusoidal” forms.

“Meandering” refers to a channel that fluctuates back and forth relativeto the overall direction of fluid flow, but that may or may not have asingle, well-defined period, or that may otherwise be irregular. Forexample, the shape of a river is “meandering.” As another example, itmay be desirable in some cases to have the period or the amplitude ofoscillation in a channelchange as a function of position, e.g.:

y(x)=A·sin(kxe ^(−mx)+θ)+D

or

y(x)=A·e ^(−mx)·sin(kx+θ)+D

“Sinusoidal” and “quasi-sinusoidal” channels thus both constitutesubsets of “meandering” channels.

In other words, for many purposes it is the alternating change in thedirection of fluid flow that is significant, not necessarily the precisemathematical function describing that direction. In practicing theinvention, it is preferred that each of the channels should have asinusoidal, quasi-sinusoidal, or other meandering shape, such that theflow of liquid through a channel tends to cause cells that are suspendedin the liquid to contact the capture elements on the channel surfacesubstantially more frequently than would be the case under otherwiseidentical conditions using an otherwise identical microdevice, but inwhich the channels were straight.

Note that a “square wave” channel or a “rectangular wave” channel is, bydefinition, not “sinusoidal.” Nor will a “square wave” or “rectangularwave” channel, in general, be considered “quasi-sinusoidal” or“meandering” within the scope of the above definitions. If 90-degreeturns are included in a channel path primarily to fold the path of thechannel and thereby to increase its length, but the turns are configuredin a manner that does not substantially enhance the frequency of contactbetween suspended cells and the channel surface (even though there mightbe some small numerical increase), then a “square wave” or “rectangularwave” channel is not considered to be “quasi-sinusoidal” or“meandering.” A sinusoidal or quasi-sinusoidal channel imparts a lateralvelocity component towards the wall, a lateral velocity that is afunction of the curvature. A square wave has a very small radius ofcurvature at a turn, and zero curvature elsewhere. Thus there areoccasional peaks in the lateral component of the velocity, but only ofshort duration. While the net effect will depend on the particulardimensions and geometry of a particular device, in general the shortduration of the peaks in the lateral component for fluid moving througha square wave channel is expected to be substantially less effective inmoving cells toward the channel walls than the smaller but longer-actinglateral component in sinusoidal channels.

What are “rare” cells is not susceptible of a precise and quantitativedefinition; qualitatively, cells are “rare” when their presence isclinically or scientifically significant or potentially significantCTCs, pathogenic bacteria), and when their numbers are vastly exceededby the numbers of other types of cells, or when the rare cells areotherwise present in very low concentrations, such that they aredifficult to detect by conventional means. Depending on the context,rare cells might, for example, be those that are present inconcentrations less than 100 per milliliter, less than 50 per mL, lessthan 20 per mL, less than 10 per mL, less than 5 per mL, less than 2 permL, or less than 1 per mL. For many purposes, less than ˜10 target cellsper mL is considered “rare” or “low abundance.”

The complete disclosures of all references cited in this specification,including the complete disclosures of the 61/053,727 priorityapplication, owe hereby incorporated by reference. Also incorporated byreference are the complete disclosures of A. Adams et al., “Highlyefficient circulating tumor cell isolation from whole blood andlabel-free enumeration using polymer-based microfluidics with anintegrated conductivity sensor,”J. Am. Chem. Soc., vol. 130, pp.8633-8641 (2008), and the associated supporting material that isavailable free of charge from pubs.acs.org; and of U. Dharmasiri et at.,“Highly efficient capture and enumeration of low abundance prostatecancer cells using prostate-specific membrane antigen aptamersimmobilized to a polymeric microfluidic device,” accepted forpublication in Electrophoresis (2009). In the event of an otherwiseirreconcilable conflict, however, the present specification shallcontrol.

What is claimed is:
 1. A method for capturing target cells from a liquidsample, comprising: (a) providing a system comprising a common input,multiple parallel channels, and a common output; wherein said channelsi) are fluidically connected to said common input and said commonoutput, ii) have an aspect ratio of 3:1 or more, and a sinusoidal orquasi-sinusoidal shape, and iii) comprise capture elements thatselectively bind molecules on membranes of the target cells; (b)hydrodynamically processing a liquid sample comprising target cellsthrough said system, wherein said channels are configured to i) causetarget cells to migrate to interior surfaces of said channels andcontact said capture elements and ii) avoid blockage in said channels.2. The method of claim 1, wherein the liquid sample is whole blood,urine, saliva, or cerebrospinal fluid.
 3. The method of claim 1, whereinthe method further comprises recovering target cells from the liquidsample with a recovery efficiency of 80% or more.
 4. The method of claim3, wherein the recovery efficiency is 85% or more, 90% or more, 95% ormore, or 97% or more.
 5. The method of claim wherein the liquid sampleis a patient sample with a known concentration of target cells.
 6. Themethod of claim 3, wherein the liquid sample is an experimental samplewith a known concentration of target cells.
 7. The method of claim 1,wherein the method further comprises recovering target cells from theliquid sample with false positives of less than 10 percent of targetcells recovered.
 8. The method of claim 1, wherein the target cells arepresent in concentrations of less than 100 target cells per milliliterof liquid sample, less than 50 target cells per milliliter of liquidsample, less than 20 target cells per milliliter of liquid sample, lessthan 10 target cells per milliliter of liquid sample, less than 5 targetcells per milliliter of liquid sample, less than 2 target cells permilliliter of liquid sample, or less than 1 target cells per milliliterof liquid sample.
 9. The method of claim 1, wherein the target cells arecirculating tumor cells, pathogenic bacteria, host cells infected withpathogen, fetal cells in maternal blood, stem cells, maternal cells inan infant or adult, erythrocytes, leukocytes, platelets, tumor cells, orcancer cells.
 10. The method of claim 1, wherein the step ofhydrodynamically processing a liquid sample through a system is at alinear flow rate of between about 2 and about 2.5 millimeters persecond.
 11. The method of claim 1, wherein hydrodynamically processingcomprises pumping.
 12. The method of claim 1, wherein the captureelements comprise antibodies, or aptamers.
 13. The method of claim 1,wherein the method further comprises the steps of: (c) removingnon-specifically bound cells from the channel surfaces, (d) releasingspecifically bound target cells from the channel surfaces, (e) countingthe number of released target cells from the liquid sample, and (f)detecting cancer.
 14. The method of claim 1, wherein the method furthercomprises the steps of: (c) removing non-specifically bound cells fromthe channel surfaces, (d) releasing specifically hound target cells fromthe channel surfaces. (e) counting the number of released target cellsfrom the liquid sample, and (f) conducting steps (a) through (e) with asecond liquid sample, and (g) determining a cancer stage.
 15. The methodof claim 14, wherein the step of determining a cancer stage comprisesdetermining a percent change in frequency in the number of releasedtarget cells sufficient to determine a cancer stage.
 16. The method ofclaim 13, wherein the step of removing non-specifically bound cells fromthe channel surfaces comprises hydrodynamic shear.
 17. The method ofclaim 13, wherein the step of releasing specifically bound cells fromthe channel surfaces comprises enzymatic hydrolysis.
 18. The method ofclaim 13, wherein the step of counting the number of released targetcells from the liquid sample comprises observing changes in theconductivity of the liquid at or near the common output.